Signal transduction is often coordinated by multi-protein complexes (Kumar and Snyder, 2002; Gavin et al., 2002; Ho et al., 2002). Constitutive interactions among proteins function in the initiation of signaling, while inducible or regulated protein-protein interactions are generally required for signal processing and propagation (Hunter, 2000). These dynamic interactions form the basis of complex regulatory circuits that determine biological function. Identifying the makeup of these circuits is a major challenge, however, because they involve multiple low affinity interactions that are controlled by dynamic post-translational protein modifications in response to multiple stimuli (Hunter, 2000; Zhu et al., 2001).
A number of non-covalent bonds form between proteins when two protein surfaces are precisely matched, and these bonds account for the specificity of recognition. Protein-protein interactions are involved in, for example, the assembly of enzyme subunits, antigen-antibody reactions, forming the supramolecular structures of ribosomes, filaments, and viruses in transport, and in the interaction of receptors on a cell with growth factors and hormones. Products of oncogenes can give rise to neoplastic transformation through protein-protein interactions. For example, some oncogenes encode protein kinases whose enzymatic activity on cellular target proteins leads to the cancerous state. Another example of a protein-protein interaction occurs when a virus infects a cell by recognizing a polypeptide receptor on the surface, and this interaction has been used to design antiviral agents.
Protein-protein interactions have been generally studied in the past using biochemical techniques such as cross-linking, co-immunoprecipitation and co-fractionation by chromatography. A disadvantage of these techniques is that interacting proteins often exist in very low abundance and are, therefore, difficult to detect. Another major disadvantage is that these biochemical techniques involve only the proteins, not the genes encoding them. When an interaction is detected using biochemical methods, the newly identified protein often must be painstakingly isolated and then sequenced to enable the gene encoding it to be obtained. Another disadvantage is that these methods do not immediately provide information about which domains of the interacting proteins are involved in the interaction. Also, small changes in the composition of the interacting proteins cannot be tested easily for their effect on the interaction.
A genetic system that is capable of rapidly detecting which proteins interact with a known protein, determining which domains of the proteins interact under physiological conditions, and providing the genes for the newly identified interacting proteins has only recently been made available. The yeast two-hybrid system currently represents the most powerful in vivo approach to screen for polypeptides that could bind to a given target protein and this invention provides a unique way of utilizing the two hybrid system for studying novel protein-protein interactions under physiological conditions.
The yeast two hybrid system described here is based on transcriptional activation. Transcription is the process by which RNA molecules are synthesized using a DNA template. Transcription is regulated by specific sequences in the DNA which indicate when and where RNA synthesis should begin. These sequences correspond to binding sites for proteins, designated transcription factors, which interact with the enzymatic machinery used for the RNA polymerization reaction.
In these systems, reconstitution into a hybrid caused by protein-protein interaction of a bait protein with a prey protein is monitored by activation of a reporter gene. Two-hybrid systems are discussed, for example, in Nandabalan et al U.S. Pat. No. 6,083,693; U.S. Pat. No. 5,283,173; U.S. Pat. No. 5,610,015; U.S. Pat. No. 5,634,463; U.S. Pat. No. 5,885,779; Klein et al. United States Statutory Invention Registration H1,892; LeGrain et al U.S. Pat. No. 6,187,535; and Rain, J.-C., et al. Nature 409, 211–215 (Jan. 11, 2001). The bait is a protein or proteins known to be involved in the pathophysiological process for which the determination is being made. The prey can be constituted of all proteins and genes expressed in cells of an affected tissue or body fluid or an election therefrom. Other methods of determining protein-protein interactions (e.g., as described in Zhu, H, et al., Science 293, 2101–2105 (2001) and as described below) can also be used. In general, the bait protein is derived from genomic DNA or a cDNA library. The cDNA library can be derived from a cell, for example, a macrophage, a cytokine activated macrophage, an endothelial cell, a muscle cell, a tumor cell or a kidney cell. Alternatively, the cDNA library can be derived from a cell treated with a drug, preferably a chemotherapeutic drug such as cisplatin.
In essence, the two putative protein partners are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively. A productive interaction between the two proteins of interest will bring the transcriptional activation domain into the proximity of the DNA-binding domain and will trigger directly the transcription of an adjacent reporter gene, for example, lacZ, giving a screenable phenotype. The transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation, as reported by Keegan et al. (1986) and Ma et al. (1987).
Transcriptional activation has been studied using the GAL4 protein of the yeast Saccharomyces cerevisiae (S. cervisiae). The GAL4 protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization, see Johnston, Microbiol. Rev., 51, 458–476 (1987). It consists of an N-terminal domain which binds to specific DNA sequences designated UASG, (UAS stands for upstream activation site, G indicates the galactose genes) and a C-terminal domain containing acidic regions, which is necessary to activate transcription, see Keegan et al. (1986), supra., and Ma and Ptashne. (1987), supra. As discussed by Keegan et al., the N-terminal domain binds to DNA in a sequence-specific manner but fails to activate transcription. The C-terminal domain cannot activate transcription because it fails to localize to the UASG, see for example, Brent and Ptashne, Cell, 43, 729–736 (1985). However, Ma and Ptashne have reported (Cell, 51, 113–119 (1987); Cell, 55, 443–446 (1988)) that when both the GAL4 N-terminal domain and C-terminal domain are fused together in the same protein, transcriptional activity is induced. Other proteins also function as transcriptional activators via the same mechanism. For example, the GCN4 protein of Saccharomyces cerevisiae as reported by Hope and Struhl, Cell, 46, 885–894 (1986), the ADR1 protein of Saccharomyces cerevisiae as reported by Thukral et al., Molecular and Cellular Biology, 9, 2360–2369, (1989) and the human estrogen receptor, as discussed by Kumar et al., Cell, 51, 941–951 (1987) both contain separable domains for DNA binding and for maximal transcriptional activation.
Recently, Rossi et al. (1997) described a different approach, a mammalian “two-hybrid” system, which uses β-galactosidase complementation (Ullmann et al., 1968) to monitor protein-protein interactions in intact eukaryotic cells. The number of genome sequences of prokaryotic as well as eukaryotic host organisms available is increasing exponentially and there is a great need for new tools directed to the functional and global study of these newly characterized complete or partial genomes.
Systems for determining protein-protein interactions which are useful herein are also described in Fung, E. T., et al, Current Opinion in Biotechnology 12:65–69 (2001) and Delneri, I., et al, Current Opinion in Biotechnology 12:87–91 (2001). Systems for determining protein interactions or activity include those described in Sakura, T., et al., Cell (1998), 573–585 and Hare, 1, et al., Nature Medicine, Vol 5, 1241–1242 (1999). These systems involve a search for an orphan receptor or ligand where readout is measured by changes in an intracellular second messenger such as calcium or G-protein activity. Other systems for determining protein interactions or activity include those described in Scherer, P., et al, Nature Biotechnology 16, 581–586 (1998). These systems involve a search for new epitopes that has been unmasked through protein-protein interaction.
Systems for determining changes in the level of protein expression are described in Fung, E. T., et al, Current Opinion in Biotechnology 12: 65–69 (2001). Systems for determining changes in the interaction between proteins and other molecules (e.g., DNA, RNA, lipids) are described in Ren, B., et al, Science 290, 2306 (2000) and in Marshall, H. and Stamler, J. S., Biochemistry 40, 1688 (2001). Methods for determining genomic interactions include methods for assaying the expression of genes in differential display, e.g., as described in Zohinhofer, D., et al., Circulation, 103, 1396–1402 (2001) and SAGE where levels of mRNA are quantified through hybridization or other means of quantification, e.g., as described in Zhang, L., et al., Science Vol. 276, 1268–1272 (1997).
Strategies for studying cellular function involved in disease states generally rely on comparison of control and disease states. A number of different proteomic and genomic strategies, including differential profiling platforms and functional assays (e.g., interaction studies) have been routinely employed for this purpose. These assays have long relied on the assumption that they accurately simulate the pathophysiological processes under investigation. However, they are carried out in “open” air and, therefore, are performed in the absence of specific and important protein modifications that are characteristic of physiological conditions, e.g., modifications that occur in the presence of nitric oxide (NO).
Nitric oxide (NO) is a ubiquitous molecule that propagates its signal through posttranslational nitrosylation of proteins (Stamler et al., 2001). Specifically, NO targets cysteine thiol and transition metal centers to regulate a broad functional spectrum of substrates, including all major classes of signaling proteins. An emerging theme in NO biology is that NO synthases (NOS) are localized within multi-protein signaling complexes where they regulate signal transduction (Stamler et al., 2001). But whether NO can directly affect protein-protein interactions transducing these signals, particularly in a disease state, has not been previously considered.
Accordingly, current screening methodologies lack a level of validation and biological significance because the actions and interactions identified using prior art methods are not causally related to the pathophysiological processes. It is therefore an object of the present invention to describe methods for identifying protein interactions under more physiological conditions. Specifically, this invention relates to the utilization of, for example, a modified yeast two-hybrid screening methodology in order to assess the possibility of NO-dependent regulation of protein-protein interactions in a cellular context.